Date: 16/03/2021

Network Pathway Analysis to analyse comorbidity of

Sleep disorders with ASD

Aditya Agarwal | Aaryan Gupta

Biological Sciences Undergraduate students
BITS Pilani BITS Pilani, Hyderabad Campus
Pilani Campus
 Aditya Agarwal 2017B1A71075H
Aaryan Gupta 2018B1A70775H

Functional Network -
Protein-protein interactions

● Protein-protein interaction
network from STRING for 67
(of total 89 curated) genes
associated with Sleep
disorders.
● Clustered using k-means
clustering with k = 6.
● Confidence level : Medium
(Score >=0.4)
● FDR stringency : Medium
(<=5%)
Legend
● Network nodes represent proteins/protein
coding gene - splice isoforms or post-
translational modifications are collapsed, i.e.
each node represents all the proteins
produced by a single, protein-coding
gene locus.
● Edges represent protein-protein
associations - associations are meant to be
specific and meaningful. Multiple edges
represent the 7 different parameters for
association.
● Color of node represents particular cluster
to which it belongs.
Pathways involved
1. Chloride channel and neurotransmitter transport - SLC6A11, SLC6A13, GABRA1, GABRA2
2. Circadian rhythm - CRY1, CRY2, PER2, PER3, CLOCK, ARNTL. BHLHE41, PER1, RORA,
NPAS2, NR1D1, CSNK1D, CSNK1E, DBP
3. DNA photolyase and period-circadian like, C-terminal - PER1, PER2, CRY1, CRY2,
CSNK1E
4. MHC class II protein complex - HLA-DRB1, HLA-DRB5. HLA-DQB1, HLA-DQA1
5. Dopamine catabolic process - MAOA, COMT
6. Catecholamine synthesis and Dopamine catabolism - SLC6A3, HNMT, MAOA, COMT,
SLC6A4.
7. EDA-ID and death inducing signalling complex assembly, tnf-alpha - TNF, TNFRSF1B,
NFKB
8. GABA A receptor activation - GABRA1, GABRA2
Pathways involved

1. Reuptake of GABA - SLC6A13, SLC6A11

2. Serotonin clearance from synaptic cleft - MAOA, SLC6A4
3. PD-1 signalling and and phosphorylation of CD3 and TCR zeta chains - HLA-
DQB1, HLA-DRB1,HLA-DRB5
4. Na+/Cl- dependent neurotransmitter transporters - SLC6A11,
SLC6A13,SLC6A3
Intra Cluster pathways

Cluster 1: TIMELESS, TIPIN, ETAA1

1. Timeless-Tipin protein complex has been reported to be important for

replication checkpoint and normal DNA replication processes
2. Mammalian TIMELESS and Tipin are Evolutionarily Conserved Replication
Fork-associated Factors
3. Human Timeless and Tipin stabilize replication forks and facilitate sister-
chromatid cohesion
4. No direct linkage between TIPIN/TIMELESS and ETAA1
Intra Cluster pathways
Cluster 2(i): Genes involved: MEIS1 BTBD9 SKOR1 TOX3 PTPRD

1. MEIS1 and BTBD9: genetic association with restless leg syndrome in end stage
renal disease
2. Periodic leg movements during sleep are associated with polymorphisms in
BTBD9, TOX3/BC034767, MEIS1, SKOR1, and PTPRD.

Cluster 2(ii): Genes involved: CPT1B CHKB PPARGC1B

3. Fatty acid oxidation (FAO)-related genes (CPT1B, CHKB, PPARGC1B)

4. CPT1B and CHKB are involved in development of narcolepsy
Intra Cluster pathways
Cluster 3: CLOCK, ARNTL, NPAS2, ARNTL2, PER1, PER2, PER3, CRY1, CRY2,
AANAT, CSNK1E, RORA, NR1D1, DBP,BHLHE41

1.Association of CLOCK, ARNTL, and NPAS2 gene polymorphisms and seasonal variations in mood
and behavior.
2.Transcription and translation of core clock components (CLOCK, NPAS2, ARNTL/BMAL1,
ARNTL2/BMAL2, PER1, PER2, PER3, CRY1 and CRY2) plays a critical role in rhythm generation.
3.Circadian-related genes: (AANAT, CLOCK, CRY1, CRY2, CSNK1E, NPAS2, PER2, PER3)
4.Associations of PER3 and RORA Circadian Gene Polymorphisms and Depressive Symptoms in
Older Adults: PER3 and RORA may play important roles in the development of depressive
symptoms in older adults.
5.Circadian Clock Genes Contribute to the Regulation of Hair Follicle Cycling: Bmal1, Clock, Dbp,
Per2, Nr1d1
6.Diseases associated with BHLHE41 include Short Sleep, Advanced Sleep Phase Syndrome,
Related pathways: Circadian rhythm and Circadian rhythm related genes.
Intra Cluster pathways

7. 17 established regulators of the cellular clock machinery that were previously

classified into 3 groups:
● E-box regulators (CLOCK, ARNTL (BMAL1), PER1, PER2, PER3, CRY1,
CRY2, BHLHE40 (BHLHE2, DEC1), BHLHE41 (BHLHE3, DEC2), NPAS2 and
ARNTL2)
● D-box regulators (DBP and NFIL3)
● RORE-box regulators (RORA, RORB, RORC and NR1D2)

8. TIMELESS: intersects both with the circadian clock and the cell cycle machinery
Intra Cluster pathways
CIITA, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB5, TNFRSF1B, TNF, NFKB1,
ADA, PRNP, IL-5, P2RY11

1. Immune system and Major Histocompatibility Complex (MHC) gene family.

a. Found to be associated with Narcolepsy
b. Immune system disorders or Autoimmune disorders found to be associated with Sleep disorders
2. Tumor Necrosis Factor proteins, NF-kb signalling
a. Proinflammatory cytokines, such as tumor necrosis factor, interleukins have been associated with
sleep dysfunction. TNFA levels are found increase with narcolepsy.
3. ADA deficiency causes grogginess
4. Fatal familial insomnia is thought to be the result of a point mutation in PRNP
5. P2RY11 commonly associated with Narcolepsy
Intra Cluster pathways
MAP2K5, HNMT, MAOA, ADRA2A, COMT, GABRA2, BDNF, SLC6A4, SLC6A3, DRD5,
HTR2A, APOE, ADORA2A, NOS1, FMR1, HCRT

1. MAP2K5 - Restless leg syndrome

2. Dopamine and serotonin transporters. Both play an important role in the sleep cycle
due to their association with melatonin. Are also involved in ASD, ADHD,
Schizophrenia. Monoamine oxidase A deficiency is also related to increase of
serotonin levels in the brain. Even COMT, ADRA2A.
3. Adrenergic receptors involved in neurotransmitter release - behaviour and sleep.
Associated with ASD, ADHD.
4. ATP consumption - High levels of Adenosine promotes sleep.
5. APOE epsilon4 allele - Alzheimer’s with sleep/wake disruption
Intra Cluster pathways
GABBR2, GABRA1, GABBR1, SLC6A13, SLC6A11, ROR1, DMPK

1. Gamma-aminobutyric acid (GABA) A and B receptors - GABA is the main inhibitory

neurotransmitter of the CNS. It is well established that activation of GABA(A) receptors
favors sleep. It has been shown that GABA(B) receptor antagonists increase brain-activated
behavioral states (waking and paradoxical sleep: dreaming stage).
2. SLC6A11, SLC6A13 are GABA transporters. Deletions in them have been associated with
ID, Epileptic seizures.
3. ROR1 also related to neural function in the brain. SNPs involved in schizophrenia.
4. The DMPK gene provides instructions for making a protein called myotonic dystrophy
protein kinase. Myotonic dystrophy type 1 (DM1) is involved in sleep disorders, including
excessive daytime sleepiness (EDS), sleep apneas, periodic leg movements during sleep,
and rapid eye movement sleep dysregulation.
Physical
subnetwork
 Date: 24/04/2021

Network Pathway Analysis to analyse comorbidity of

Sleep disorders with ASD
Post Midsem
Aditya Agarwal | Aaryan Gupta
Undergraduate students
BITS Pilani BITS Pilani, Hyderabad Campus
Pilani Campus
79 genes curated from
literature, backed by 1002 genes from SFARI
experimental or statistical (universally accepted).
findings. Includes genes
from recent publications.

15 genes common
between the two lists.
Combined interactome of 15
common genes b/w ASD
and sleep with Genes with
direct links (through pathway
interactions) to them.
Genes with direct links
(through pathway
interactions) to the 15
common genes b/w ASD
and sleep.
1. 4 genes with a link to parkinson’s disease which is
also a neurodegenerative disorder even though being
prevalent in older age.
2. PARK2 and BTRC are involved in ubiquitination to tag
excess proteins.
3. DDC produces dopamine and other neurotransmitters
while MAO-B degrades them. Both regulate mood.
4. DVL3 and CSK1G1 are involved in wnt signalling, cell
proliferation, growth.
5. CTNNB1 is important for cell growth, specialization.
Syndrome with developmental delay.
1. ADK - This sub pathway is part of the pathway AMP
biosynthesis via salvage pathway, which is itself part
of Purine metabolism.
2. ADCY5 converts ATP, into cAMP, which is used in
many cell signaling processes.
3. ADORA3 encodes a protein that belongs to the family
of adenosine receptors, which are GPCRs that are
involved in intracellular signaling pathways and
physiological functions.
4. RORB Gene encodes family of orphan nuclear receptors
that are believed to play a role in circadian rhythm and
bone metabolism.
5. NDUFA5 is part of NADH dehydrogenase subunit. Not
significant to cluster.
1. DRD1 and DRD2 encodes the D1,D2 subtypes of the
dopamine receptor. These subtypes are the most
abundant dopamine receptors in the central nervous
system and the brain.
2. CAMK2A and CAMK2B belong to the
serine/threonine protein kinases family, and to the
calcium /calmodulin-dependent protein kinases
subfamily. Involved in mental retardation.
3. GNAS and GNAI1 produce alpha subunit of G
proteins which function as transducers in numerous
signaling pathways controlled by GPCRs.
4. GNAI1 inhibits adenylate cyclase while ADCY3
produces it and its function is to catalyze cAMP.
1. PlCB1 plays a crucial role in the initiation of the
genetic program responsible for muscle differentiation
and osteogenesis. Diseases associated with PLCB1
include Developmental And Epileptic Encephalopathy
and Malignant Migrating Partial Seizures Of Infancy.
2. PRKCA polymorphism changes the neural basis of
episodic remembering in healthy individuals.
3. PRKCA and PRKCB involved in MAPK signalling.
4. The ITPR1 gene provides instructions for making a
protein that is part of a channel that controls the flow
of positively charged calcium ions within cells
5. AVPR1A and AVPR1B are vasopressin receptors,
GPCRs.
6. OXT is also involved in GPCR signalling and
Oxytocin signalling pathway.
1. The GABA transporter that is made by the gene
SLC6A1 is essential in removing extra GABA from the
spaces between two neurons.
2. SLC1A1 and SLC1A2 produce EAAT2 and GLT1,
both being involved in glutamatergic synapse.
3. The CACNA1A and CACNA1B genes belong to a
family of genes that provide instructions for making
calcium channels. They play a vital role in the
communication between neurons in the brain.
4. ATP2B2 plays a critical role in intracellular calcium
homeostasis.
5. STX1A encodes a member of the syntaxin
superfamily. Syntaxins are nervous system-specific
proteins implicated in the docking of synaptic vesicles
with the presynaptic plasma membrane.
1. EIF4E encodes for translation initiation factor and is
involved in MAPK signalling, interferon gamma
signalling pathways.
2. The protein encoded by EIF4G1 is a component of
the multi-subunit protein complex EIF4F.
3. In the CYFIP1-EIF4E-FMR1 complex this subunit is
an adapter between EIF4E and FMR1. Promotes the
translation repression activity of FMR1 in brain
probably by mediating its association with EIF4E and
mRNA. Mutations cause deficit in reasoning.
15 common genes b/w
ASD and sleep.
1. NR1D1, CSNK1E, RORA, NPAS2, PER1, PER2, are all
part of the sleep interactome and are involved in Circadian
rhythm pathway
2. NR1D1 negatively regulates the expression of core clock
proteins.
3. PER1 and PER2 are important for both the maintenance of
circadian rhythmicity and entrainment to light cues.
4. Transcription of NPAS2 is enhanced by the
RORA:Coactivator complex
5. NR1D1 binds the promoter of the NPAS2 gene and
recruits corepressors to repress transcription.
6. RORA, NR1D1 are some of the genes implicated in
metabolism and PER2 in sleep homeostasis.
1. Synaptic serotonin is primarily maintained by metabolizing
enzyme MAOA and serotonin transporter (SLC6A4) which is
involved in Serotonergic synapse pathways and helps in
reuptake of the neurotransmitter. This is known to control
emotion, mood and drive.
2. The SLC6A3 and SLC6A4 genes are members of a class of
neurotransmitter transporters for the release, re-uptake and
recycling of neurotransmitters in synapses.
3. SLC6A3 and MAOA are found in cocaine and amphetamine
addiction and Dopaminergic synapse pathways. MAOA
encodes mitochondrial enzymes which catalyze the oxidative
deamination of amines, such as dopamine, norepinephrine,
and serotonin. Deficiencies in MAOA activity have been
identified to correlate positively with aggressive behaviour.
4. FMR1 codes for fragile X mental retardation protein, or
FMRP which is essential for normal cognitive development
and female reproductive function. FMRP and FXR2P
together can increase PER1 or PER2-mediated BMAL1-
Neuronal PAS2 (NPAS2) transcriptional activity. FMR1 is
required for the presence of rhythmic circadian behavior in
mammals.
1. ADA produces an enzyme called adenosine
deaminase, which converts adenosine to inosine.
Adenosine is important for sleep regulation and its
levels increase the longer the person stays awake.
2. ADORA2A is an adenosine receptor, Upon binding
with ADORA2A adenosine induces sleep,
especially when present at high doses, so the
natural build up of adenosine in the brain during
the day is used to promote sleep, allowing the
brain to recover its energy stores.
3. Mutations in CHKB have been found to result in
mitochondrial deficiencies and associated
disorders resulting in major disorders in the
function and structure of the mitochondria,
example in narcolepsy.
What do we infer?
Tye et. al (2019) proposed the possible mechanisms of the interplay between ASD and Sleep disorders - The circadian
rhythm is dysregulated due to alterations in neurophysiology and neurochemistry. Neurochemistry factors involved
neurotransmitters GABA, melatonin and serotonin.

The DDC gene is majorly involved in production of melatonin, dopamine and serotonin and mutations have been
associated with ASD. The DDC gene in our interactome has direct connections with MAOB gene which metabolizes
dopamine. MAOB has not yet been reported as a candidate gene for Sleep disorders. However, MAOA has been
implicated in sleep disorders. The difference between MAOB and MAOA is the difference in substrate selectivity. MAOA
more actively metabolizes serotonin, epinephrine and norepinephrine. MAOA is an important gene in the sleep
interactome cluster with connections to circadian rhythm genes, clock genes and dopamine transporter genes. MAOA
connects to AANAT which is the prime gene for converting serotonin to melatonin and inducing sleep. Interestingly,
studies also suggest MAOA/MAOB are prime candidates for the investigation into the role of DNA methylation in mental
disorders.

The metabolism of neurotransmitters is therefore an extremely important aspect to explain the comorbidity of sleep
disorders with ASD and the interactome(s) suggest the same and help to identify the possible links at the genetic level.
What do we infer?
Neurotransmitters play a critical role in neural communication, influencing everything from involuntary
movements to learning to mood. But, this is incomplete without the action of neurotransmitter transporters at
the synaptic junctions that control the level of neurotransmitters at the synapse.

SLC1A1 and SLC1A2 mutations/deletions are associated with Autism and their function is related to
glutamate transporter protein production. SLC6A1, SLC6A3 and SLC6A4 produce GABA, dopamine and
serotonin transporters and therefore, as expected, the latter are implicated in both ASD and sleep. By
examining the interaction between SLC6A1 and SLC1A2 in the interactome, it is found that they are co-
expressed together. There is therefore, close interplay between the transporter genes.

What this information suggests is that alongside neurotransmitters and the genes that produce and
metabolize them, the proper functioning of their synaptic transporters is important to regulation of the
neuronal circuitry and communication. Disruption in their expression can therefore lead to disruption of the
circadian cycle which is maintained by neurochemistry of these neurotransmitters hence indicating a
possible connecting link between ASD and sleep.
What do we infer?
Adding to the connection between neuronal communication, ASD and sleep is the role of
intracellular signalling pathways, specifically the cAMP signalling pathway and MAPK signalling for
cellular communication.

Multiple genes from the interactome are involved either directly to the cAMP and MAPK signalling
pathway or indirectly through the production of GPCRs that assist in signalling and are present at
the cell membrane. GPCRs respond to the presence of neurotransmitters/growth factors/stress
near the membrane and accordingly control the intracellular signalling pathways.

Attenuation in cAMP signalling in the hippocampus has been associated with sleep deprivation.
While MAPK signalling has been shown to be perturbed in Autism. It can be seen that intracellular
signalling is equally important to both ASD and Sleep disorders and could be an important factor to
explain comorbidity of sleep with ASD in patients with attenuated signalling pathways.
 ASD
MAPK Signalling pathway

Dopamine
Glutamate

Melatonin
Serotonin

cAMP Signalling pathway

SLC transporters for
Sleep disorders Glutamate, Serotonin
and Dopamine at the
Synaptic junction.
What do we infer?
1.ADA gene produces an enzyme called adenosine deaminase, which converts adenosine to inosine.
Adenosine is important for sleep regulation and its levels increase the longer the person stays awake. Apart
from sleep, it is also reported that reduced ADA activity is seen in autistic children. The neuromodulator
adenosine has been shown to affect certain ASD comorbidities and symptoms, such as epilepsy, impairment
of cognitive function, and anxiety. Adenosine is both directly and indirectly responsible for regulating the
development of oligodendroglia and myelination.

3.. Recent evidences suggest that white matter and myelin abnormalities are more relevantly involved in
ASD pathophysiology.

5. Magnetic resonance imaging (MRI) and DTI studies show that white matter disruption occurs in brain
regions of children with ASD.

7. Anisotropy value was reduced in the white matter in children and adolescents with autism which
suggests that white matter disruption between brain regions may cause impaired social cognition in autism.

8. The broad actions of the adenosinergic system on neuroprotection, neuroinflammation, modulation of

neurotransmissions, and regulation of glial function are involved in the core behavioral symptoms in ASD.
Therefore, manipulating adenosine signaling pathways and affecting oligodendrocyte activities, can be
What do we infer?
1. FMR1 codes for fragile X mental retardation protein, or FMRP which is essential for normal cognitive
development. FMRP deficiency may cause not only degeneration of dendrites and synapses, but also
ROS overproduction, elevated levels of intracellular ROS have been implicated in the occurrence of
oxidative stress and subsequent apoptotic cell death that causes brain damage, neurotoxicity and
neurodegeneration

2. FXS (Fragile X Syndrome) is the most common genetic cause of autism. FXS is caused by a mutation
of the fmr1 gene on the X chromosome. In FXS, abnormalities in dendritic spines are induced by the
silencing of the fmr1 gene, and the resultant absence of FMRP may alter the morphology and synaptic
number of dendritic spines. FMRP is associated with the regulation of synaptic structure and function.
Thus, the loss of FMRP in FXS due to fmr1 gene silencing may imply the presence of
neurodevelopmental abnormalities.

The overexpression of FMRP increases PER1- and PER2-mediated BMAL1 –NPAS2 transcriptional
activity, which suggests that FMRP is an essential component involved in the regulation of rhythmic
circadian behaviors.
● Several studies have suggested that melatonin is a very powerful free radical scavenger and
antioxidant.
● Clinical studies have reported decreased levels of melatonin in the blood of individuals with FXS
and ASD
● Despite the fact that sleep is one of the major concerns for families having a child with ASD, this problem was often
considered as an epiphenomenon and therefore did not catch the attention of the scientific community.
● However, recent results showing abnormal melatonin synthesis, as well as an efficacy of melatonin therapy for sleep
problems observed in ASD, may change this initial disregard from a possible key role of the clock and circadian
regulations in ASD.
Melatonin is one of the factors that sets the internal clock to a 24-hour cycle and therefore is crucial for appropriate
regulation of the sleep/wake (S/W) cycle

Interviews with the parents of patients with ASD revealed that more than 70% of patients had delayed
development of the circadian S/W cycle by at least 5 months. This problem persists through childhood because 50–
80% of children with ASD show highly significant increased sleep latency and nocturnal awakenings
●
For that reason, the early detection of a melatonin deficit in children may greatly improve the efficacy of melatonin
treatment in ASD.
https://www.mdpi.com/1422-0067/18/5/938/htm